[3-oxo-20-hydroxy-1, 4, 17(20)-pregnatrien-16-yliden] acetic acid lactone, and intermediates therefor



p C Patented Jul j ii can be prepared from naturally existingkryptogenin, ac- 3,043,834 cording to the method of the presentinvention. [3-0X0-20-HYDR9XY PREGNATRIEN- Accordingly, a primary objectof the present invention LACTONE, AND is to provide a utilization methodfor the said natural Kik'uo lgamshi Osaka Japaslgssignor to Shionogi &steroid. Another object of the present invention is to PIOVl e a synetic me o ort e preparaion o a ren- Co. Lt d. Osaka Japan th th df t f dNo Drawing. z Dec 11 1961, sen 158,529 ocortical hormones. Another obect of the lnvention 18 Claims priority, application Japan Dec. 31, 1958to provide the A -3-oxo stero1dal lactone, corresponding 13 Claims. (Cl.260--239.55) to Formula I, intermediates therefor and processes fortheir preparation. The present invention relates to steroidal lactonesand The method of the present invention can be illustrated intermediatesthereof, and more particularly to [3-oxoby the following scheme:

: To I I onto 0 0R 01120 0 OR R0 no RO- l I I H H H (II) (III) (IV)RIIIO R O =0 00 =0 omo 0 on OHQ R" c1120 0 0R" no 1 R0 RO- no o- I(VIII) (IX) (I) -hydroxy-1,4,1-7(20)-pregnatrien-l6-yliden]-acetic acidwherein each of R, R and R'" represents hydrogen or lactone andintermediates thereof. a lower acyl group having from 1 to 10 carbonatoms The Y Y )-P g atrien 16- such as formyl, acetyl, propionyl,butyryl, valeryl, ocyliden] -acetic acid lactone, according to thepresent intanoyl, decanoyl and the like, and R" represents hydro-Ventiofl, corresponds to the formula! gen or a lower alkyl group havingfrom 1 to 10 carbon atoms such as methyl, ethyl, propyl, butyl, pentyl,octy-l,

To decyl and the like. The scheme should be understood as I showing thesubstantial conversion; the acyl radical, e.g. the 3-acyl radical, insome compounds shown therein may be changed to the hydroxyl radical,depending on the reagent used, in the course of reaction in some steps.However, the resulting hydroxyl compound can be easily changed into thecorresponding acyl compound by a conventional esterification procedure.

(I) Kryptogernn is known to occur in significant amounts The A1,4-3-oxosteroidal lactone I is a form of synin several species of MexicanDiscorea, and it can be thetic adrenocortical hormone and can beproduced from easily changed through kryptogenin diacylate (RE.5,6-dihydrokryptogenin diacylates, which are known and Marker et al.:J.A.C.S., 69, 2198 (1947)) into 5,6-dihy- 3 drokryptogenin diacylate (H.Hirschmann et al.: Tetrahedron, 3, 251 (1958)). In the presentinvention, 5,6-dihydrokryptogenin d1- acylate, corresponding to FormulaII, is used as the starting material. Firstly, it is subjected toaddition with haloacetic ester andzinc, followed by hydrolysis withacid. The addition reaction is conveniently carried out by heating the5,6-dihydrokryptogenin diacylate II with haloacetic ester and zinc in asolvent, preferably in the presence of a catalyst. As the haloaceticester, there may for example be employed chloroacetic ester such asmethyl chloroacetate, ethyl chloroacetate or propyl chloroacetate,bromoacetic ester such as methyl bromoacetate, ethyl bromoacetate,propyl bromoacetate or butyl bromoacetate, or iodoacetic ester such asmethyl iodoacetate, ethyl iodoacetate or propyl iodoacetate. Althoughany one of these illustratively disclosed haloacetic esters can be used,a bromoacetic ester is actually preferred. It is also preferred that thezinc be pure and that it have a fresh, clean surface. Zinc dust, zincfoil, granulated zinc and mossy zinc may equally well be used, but anyof the forms of zinc employed should be purified by washing with asolvent. Usually, the zinc is used in the reaction after washing it withdilute acid, water, alcohol and acetone in turn and drying in vacuo. Asthe solvent, in which the reaction is carried out, there may be usedbenzene, toluene, xylene or the like. Although the use of a catalyst isnot always necessary, it may be helpful in initiating the reaction touse a few crystals of iodine, a little amalgamated zinc or a littlealkylmagnesium iodide, when difllculty is encountered. Theintermediarilyformed addition complex, corresponding to the followingwherein R, R and R" have the hereinbefore-stated meanings and X ishalogen such as chlorine, bromine or iodine is treated with a diluteacid such as dilute hydrochloric acid or dilute sulfuric acid at roomtemperature (15 to 30 C.) or while ice-cooling to yield a cyclic ethercompound, corresponding to Formula III.

Secondly, the thus-obtained cyclic ether Compound III is subjected tocyclization by treatment with alkali, followed with acidifying. Thereaction can conveniently be carried out by allowing the cyclic etherIII to stand at roomtemperature, or by heating the cyclic ether CompoundIII, with an alkali such as sodium hydroxide, potassium hydroxide,sodium carbonate, sodium bicarbonate, potassium carbonate, potassiumbicarbonate and the like, and then treating the reaction product with amineral acid such'as hydrochloric acid, sulfuric acid and the like. I

Thirdly, the resulting spirostane, corresponding to Formula IV, issubjected to pseudomerization (conversion into the pseudo form). Thepseudomerization reaction may be carried out by treating the spirostaneIV under conditions providing an acylonium ion, i.e. by heating the saidspirostane IV with an acid anhydride in the presence of pyridinehydrochloride or a Lewis acid as a catalyst. As the Lewis acid, theremay for example be employed hydrochloric acid, acetyl chloride, aluminumchloride, p-toluenesulfonyl chloride, zinc acetate or trichloroaceticacid. Generally, it is preferred to execute the reaction in the presenceof pyridine hydrochloride.

Fourthly, the resulting pseudo form compound, corresponding to FormulaV, is subjected to oxidation with an oxidizing agent. As the oxidationagent, there may advantageously be used chromic acid-acetic acid,hydrogen peroxide-acetic acid, potassium permanganateacetic acid, ozoneand the like. The reaction conditions for the oxidation may be selectedaccordingly to the general knowledge in the use of these oxidizingagents.

Fifthly, the thus-obtained pregnane compound of Formula V1 is subjectedto elimination of the substituent at the 16-position. The reaction canbe carried out by the treatment of the pregnane Compound V1 with alkali.The reaction may proceed in two steps, i.e. hydrolysis at l6-positionand dehydration between the 16- and 17- positions, to yield thel6-pregnene of Formula VII. Sometimes, the laconization reaction followsimmediately to give the 3-hydroxylor acyloxy steroidal lactone ofFormula VIII. Whether or not this takes place depends on the reactionconditions, e.g. alkali employed. For instance, the treatment of thepregnane Compound VI with alkali metal hydroxide for a short time givesthe l6-pregnene VII, while treatment with alkali metal bicarbonate inlower alkanol gives the 3-hydroxyl or acyloxy steroidal lactone VIII.Generally speaking, the said lactonization reaction may take place whenthe alkalinity is relatively strong. The conversion of the l6-pregneneVII into, the 3-hydroxyl or acyloxy steroidal lactone VIII can be easilyaccomplished by treatment with an acid such as hydrochloric acid,sulfuric acid or acetic acid or also with an acid 'anhydride, e.g. inthe presence of pyridine.

Sixthly, the thus-obtained 3-hydroxyl or acyloxy steroidal lactone VIIIis subjected to oxidation of the hydroxyl radical at the 3position tothe oxo radical, if the substituent at the 3-position' is an acyloxyradical, with the previous conversion of the acyloxy radical into thehydroxyl radical. The previous conversion can be easily executedaccording to a conventional hydrolyzing procedure, e.g. treating withacid. The oxidation reaction may be carried out by'treating the3-hydroxyl steroidal lactone VIII with an oxidizing agent. As theoxidizing agent, there may be advantageously used aluminum trialkoxideand ketone or chromic acid. In the former case, the reaction may becarried out by refluxing the 3-hydroxyl steroidal lactone VIII withaluminum trialkoxide, such as aluminum triisopropoxide, aluminumtriisobutoxide or aluminum tri-t-bu-toxide, and ketone, such as acetone,methyl ethyl ketone or cyclohexanone, preferably in an inert organicsolvent such as benzene, toluene or xylene. In the latter case, thereaction may be carried out by treating the 3- hydroxyl steroidallactone VIII with chromic acid or chromic acid-sulfuric acid in an inertsolvent such as acetone or glacial acetic acid at a lower temperature.

Seventhly, the resulting 3-oxo steroidal lactone, corresponding toFormula IX, is subjected to dehydrogenation. The reaction may be carriedout by refluxing the 3-oxo steroidallactone IX with selenium dioxide inan inert solvent such as dioxane, acetic acid, benzene, methanol,ethanol or butanol. As the result of the reaction, the objective A-3-oxo steroidal lactone I is obtained as a mixture with the diselenidecompound, corresponding to the following formula:

I ll l The A -3-oxo steroidal lactone I can be easily separated from thesaid reaction mixture according to the c0nventional purificationprocedure, e.g. fractional recrystallization or chromatography.

Although the method of the present invention is hereinbefore illustratedstep by step, these steps may be executed successively without theisolation of the product in each step.

'llhe final product of the present invention [3-oxo-20-hydroxy-1,4,17(20) pregnatrien 16-yliden] acetic acid lactone of FormulaI, is a form of synthetic adrenocortical hormone, and has physiologicalactivities. When the A 3-oxo steroidal lactone I is administeredtogether with desoxycorticosterone (DOCA) to the adrenoprival mouse,regulation of sodium and potassium metabolism is not affected, but waterretention is selectively increased. The A -3-oxo steroidal lactone I isuseful for the particular treatment of adrenal cortical insufficiency,and from the fact mentioned above it appears that some effectiveantidesoxycorticostero-ne agents may exist in the group of analogouscompounds. In fact [3B,20-dihydroxy-5a-l7 (20)-pregnen-l6-yliden]-aceticacid lactone of Formula V111 controls the activity ofdesoxycorticosterone completely, when administered together withdesoxycorticosterone in the amount of 500 times of the latter.

Thus, the process involved in the present invention has significantutility with respect to the synthesis of physiologically activesteroids, and the thus-obtained products are also useful adrenocorticalhormones or intermediates.

The following examples represent presently-preferred embodiments of thepresent invention, but it is to be understood that the examples aregiven by Way of illustration only and not of limitation. Theabbreviations used in these examples are intended to have the followingmeans: g.', gram; mg., milligram; ml., milliliters; C., degreescentigrade; and M.P., melting point.

Example 1 I jo 0113000: l o

CH O'OO I 0 0113000- OZnBr l --c1120 oocnn orncoo l H g. of5,6-dihydrokryptogenin 3,26-diacetate are dissolved in 60 ml. of drytoluene and 40 ml. of dry benzene, and 4 g. of granulated zinc "areadded. Then the mixture is boiled in an oil bath to eliminate moisture,and cooled. To the mixture, 10 g. of ethyl bromoacetate and a fewcrystals of iodine are added, and the whole heated mildly with stirringuntil reaction starts. As soon as reaction sets in, the oil bath isremoved and the mixture is cooled with water so that the reactionproceeds mildly. After refluxing mildly for 1 hour more, the mixture ispoured into dilute sulfuric acid with ice-cooling to hydrolyze theaddition complex. The organic layer is separated and the aqueous layeris extracted with two m1. portions of chloroform. The extracts arecombined with the previously separated organic layer, washed with dilutesodium carbonate solution and water, dried over sodium sulfate, and thesolvent removed under reduced pressure. The residue is dissolved in 300ml. of methanol, and 30 g. of 20% potassium bicarbonate solution areadded, and the solution refluxed for 5 hours in an oil bath. Afterremoving the methanol under reduced pressure, water is added to theresidue and the resultant precipitates are collected by filtration,washed with Water and chloroform thoroughly, and the filtrate extractedwith chloroform. The aqueous layer is combined with the previouslyobtained precipitates, and acidification effected with dilutehydrochloric acid. The thus-precipitated crys tals are collected byfiltration, washed with water and dried. Recrystallizing from methanol,there are obtained 4.6 g. of 3fl-hydroxy 5u,225 spirostan-16a-aceticacid (tigogenin-16a-acetic acid), M.P. 254 to 255 C. (dec.). [a] 52.6(14) (in dioxane),

LR. 1123i? 3520, 984, 902, 888, 865 cm.-

(888 cur- 902 cm) Analysis.--Ca1cd. for 0 91 14 0 C, H, 9-77. Found: C,73.10; H, 9.73.

In conventional manner, the following compounds are obtained from theabove product:

O I omooocn,

Methyl 3 3-hydroxy-5a,22 8-spirostan-l6ot-acetate: M.P. 194 to C. [111--44.9 (i2) (in chloroform). Methyl3fi-acetoxy-5a,22fi-spirostan-16u-acetate: M.P. 167to 168 C. [M 63.2(:2") (in chloroform).

Example 2 crnooocrn Cacao- 0 l oniooon HO I To 50 ml. of aceticanhydride, 5 g. of methyl 3,8- acetoxy-Sa,22B-spirostan-16a-acetate and2.2 g. of pyridine hydrochloride are added and the mixture heated withrefluxing on an oil bath for 5 hours. After concentration under reducedpressure, the reaction mixture is extracted with ether. Theextract isWashed successively with water, sodium bicarbonate and water and driedover sodium sulfate. Removal of ether yields crude crystals of methyl3fi,26-diacetoxy-5a-20(22)-furosten-16:x-acetate.

The so-obtained crystals are hydrolyzed with potassium hydroxide inmethanol and the resulting product is recrystallized from ethylacetate-methanol to give 35,26-dihydroxy-5a-20(22)-furosten-16a-aceticacid as crystals melting at 201 to 202 C. (dec.). Yield 85%. LR. 11 max3290(OH), 2685-27l5(COOH), 1700 (Vinyl ether) cmr Analysis.-Calcd. for CH O C, 73.38; H, 9.77.

Found: C, 73.34; H, 9.70.

Example 3 CHaG O O cmcoo- =0 00 I lomooocn,

GHaOOO 1.032 g. of crude methyl 3fl,26-diacetoxy-5a-20(22)-furosten-16a-acetate are dissolved in 10 ml. of glacial acetic acid. Tothe solution, 9.1 ml. of a solution of N chromic acid in acetic acid areadded under icecooling, and the whole allowed to stand for 15 hours.Then 4 ml. of methanol. are added to the reaction mixture to decomposeexcess of chromic acid, and the mixture extracted with ether. Theextract is washed with water, the solvent removed and the residuetreated with ether-petroleum ether. The crystallized substances (being aby-product) is removed and the mother liquor is heated with 50 ml. ofmethanol, 2 ml. of water and 1.5 g. of potassium bicarbonate whilerefluxing for 40 minutes. After cooling, the resulting mixture isextracted with chloroform and the extract is washed with water.Evaporation to dryness gives 610 mg. of residue. The residue isdissolved in benzene and is then chromatographed on 4 g. of Florisil(activated magnesium silicate) to yield 474 mg. of crude crystals. Thecrude crystals are recrystallized from acetone-petroleum ether to give[35,20 dihydroxy 5a 17(20) pregnen l6 yliden] acetic acid lactone ascrystals melting at 198 to 199 C.

Example 4 p} CHzCOOCH;

orrrooo I GHaCO 0 To a solution of 1.014 g. of methyl 3 B,26-diacetoxy-5a-20(22)-furosten-1Got-acetate in 10 ml. of glacial acetic acidare-added 10 ml. of glacial acetic acid and 10 m1. of 35% hydrogenperoxide. The mixture is allowed to stand at 20 C. for 6 hours, and thensubjected to the treatment for decarboxylation as described in Example 3to give 383 mg. of [3fl,20-dihydroxy-5a-l7(20) pregnenl6-yliden1-aceticacid lactone.

Example 5 omooo a I -0111000011,

' CHaOOO =0 00 j I 0 I I lomcoocn,

CHzQOO To a solution of 1.031 g. of methyl 3,8.26-diacetoxy-SOL-20(22)-flll'OSt6D-16ot-3C6tfit6 in 45 ml. of glacial acetic acid isadded a solution of 467 mg. of potassium permanganate in 33 ml. of 60%acetic acidwith stirring while ice-cooling. Then the reaction mixture isallowed to stand at 20 C. for hours and sub jected to the treatment fordecarboxylation as described in Example 3 to give 293 mg. of

yliden] -acetic acid lactone.

Example 6 0113000: l cmoooom omooo I Into 1 g. of methyl3,l,26-diacetoxy-5u-20(22)-furosten-16a-acetate in 15 m1. of chloroform,ozone is introduced while cooling with Dry Ice-acetone. The mixturebegins to show a pale blue color within 1.5 hours. The

chloroform is then removed at a lower temperature. The residue isdissolved in 15 ml. of glacialacetic acid, 1.3 g. of zinc powder addedwhile cooling, and the whole allowed to stand with occasional stirring.After the reaction mixture becomes negative in iodo-zinc-starch paper,Water is added and the treatment as described in Example 3 fordecarboxylation carried out to give 362 acetic acid lactone.

. Example 7 water, dried and treated with acetone.

' (c.=0.990 in chloroform).

In 25 ml. of methanol, there are dissolved 1.903 g. of the oxidationproduct obtained as an intermediate in Example 3. To the resultantsolution are added 2 g. of potassium hydroxide, ml. of water andlO ml.of methanol, and the whole heated with refluxing for 3 minutes. Thereaction mixture is diluted with Water, and then dilute hydrochloricacid is added to acidity. The resultant crystalline substance iscollected by filtration, washed with The acetoneextract ischromatographed on 20 g. of silica gel. The

Y eluates with chloroform-acetone (95:590:10) are crystallized firomacetone to give 228 mg. of 3B-hydroxy-20-oxo-5a-16(l7)-pregnen-16-acetic acid. MP. 173 to 174 C. (recrystallizedfrom dichloromethane-acetone).

- 1.1111221? 3440, 3206, 1729, 1687, 1660, 1e44, 1611 cmr U.V. im 253 mp(617560).

Example 8 In a mixture of 20 mlfoftoluene and ml. of cyclo hexanone,there are dissolved 488 mg. of[35,20-dihydroxy-5a-17(20)-pregnen-16-yliden]-acetic acid lactone. IFrom the solution,- about 10 ml. of thesolvent are removed. Totheresulting solution, 400mg. of aluminum triisoprop'oxide are added andrefluxed forl hour. The reaction mixture is poured into 100 ml. of, anice-cooling 2% hydrochloric acid. The organic solvent layer isseparated, washed with water and dried. Removing the solvent, theresidue is extracted with chloroform. The extract is washed with waterand dried, and the solvent is removed. The resulting substance iscrystallized out from ether-petroleum ether and recrystallized fromacetone 'to afford 230 mg. of [3-oxo -20-hydroxy-5a-17(20)-preg- MP. 242to 245 C. [a] -0.9 :2)

nen-16-yliden]-acetic acid lactone. (gradually coloured from 225 C.)

Example 9 A solution of 26.72 g. of chromic acid in 23 ml. of cone.sulfuric acid is diluted with water to make 100 ml. To a solution of 475mg. of [3,8,20-dihydroxy-5a-17(20% pregnen16-yliden]-acetic acid lactonein 50 ml. of ace: tone, 0.46 ml. of the said chromic acid-sulfuric acidsolution .is added with stirring, and the further stirring is continuedfor 5 minutes. The reaction mixture is poured into 300 m1. of water andthen extracted with chloroform. The extract is washed with water anddried, Removing the solvent, the residue is recrystallized from acetoneto afford 286 mg. of [3-oxo-20-hydroxy-5a-17(20) -pregnen-16-yliden1-acetic acid lactone as crystals melting at 242' to 245 C.

Example 10 filtrate is concentrated in 'vacuo, and the residue isdissolved in ethyl acetate. The resulting ethyl acetate solution iswashed in order with potassium bicarbonate, water, freshly preparedice-cooling ammonium hydrogen sulfide, ice-cooling dilute ammonia water,water, dilute hydrochloric acid and water, and dried. Removing thesolvent,

I the residue (285 mg.) is chromatographed on 8 g. ofaluylidenjl-acetice acid lactone.

mina. Recrystallization of 124 mg. of the benzene-chloroform (921 to6:4) eluates from acetone-hexane affords 54 mg. of[3-oxo-20-hydroxy-1,4,17(20)-pregnatrien-l6- M.P.- 265 to 267.5" C., [a]-16.2(:2) (c.=1.016% chloroform).

LR. 11%;? 1730, 1712, 1662, 1619, 1605, 1586 cm. U.V wig 3 243 m(ezl7000); 301 m (ez7000) Analysis.Calcd. for C H O C, 78.82; H, 7.48.Found: C, 78.71; H, 7.48.

Recrystallization of 70 mg. of the chloroform eluate from acetone-hexaneaflords 20 mg. of diseleno-2,2'-bis- [(3 oxo20-hydroxy-1,4,17(20)-pregnatrien-16-yliden)- acetic acid lactone]. M.P.306 C. (decomposition). [a],;, -323.0(- 5) (c.=0.651 chloroform). I LR.1122i? 3300-3580 (OH), 1728, 1710, 1695, 1641,1620, 1586 emf}. U.V. xiii1 246, m (ezllOOO); 260 mu (6:11100); 301 m (616900);

Analysis.--Calcd. for C43H500 Se2'3H20: C, H,

6.17. Found C, 60.84; H, 6.18.

While the foregoing examples are directed to simplest 13 and presentlypreferred specific embodiments, more complex embodiments wherein themoieties corresponding to R, R" and R' contain up to carbon atoms maymutatis mutandis be similarly prepared and used.

This application is a continuation-in-part of copending application,Serial No. 861,212, filed December 22, 1959 (abandoned since the filingof the present application).

What is claimed is:

l. [3-oxo 2O hydroxy 1,4,17 (20) pregnatrien 16- yliden]-acetic acidlactone.

2. [3-0X0-20-hydroXy 5a-l7(20)-pregnen 16 yliden]- acetic acid lactone.

3. A compound of the formula wherein R is a member selected from thegroup: consisting of H and alkylcarbonyl, the alkyl moiety of the lattercontaining from 1 to 10 carbon atoms.

4. [3,8,20-dihydroary6a-17(20) pregnen 16 yliclen]- acetic acid lactone.

5. [3B-alkylcarbonyloxy-20-hydroxy c 17 (2-0)-pregnen-16-yliden1-aceticacid lactone, wherein the alkyl moiety contains from 1 to 10 carbonatoms.

6. [3p-acet0xy-2'O-hydroxy 50c 17(20) pregnen-l6- yliden]-acetic acidlactone.

7. 3/3-hydroxy-5u,22fl-spirostan-l6a-acetic acid.

8. Methyl 35-hydroxy-5a,22fi-spirostan-16 x-acetate.

9. Methyl 3fl-alkylcarbonyloxy 511,225 -"spirostan-l6aacetate, whereinthe alkyl moiety contains from 1 to 10 carbon atoms.

.10. Methyl 3B-acetoxy-MQZfl-spirostan-l6a-acetate.

11. 3fl,26-dihydroxy-5a-20(22) furosten 16a acetic acid.

12. Methyl 3p,26-diacetoxy-5a-20(22) furos-ten cacetate.

13. 3p-hydroXy-20-oxo-5u-16(17)-pregnen l6 acetic acid. 6

References Cited in the file of this patent Noller: Chemistry of OrganicCompounds, 2nd ed., 1957, W. B. Saunders 00., Philadelphia, P-a., p.236.

1. (3-OXO - 20 - HYDROXY - 1,4,17(20) - PREGNATRIEN - 16 YLIDEN)-ACETICACID LACTONE.